This invention concerns scintillation proximity tests, that is to say assays or other experiments involving the scintillation proximity principle.
Current scintillation proximity assay (SPA) technology involves the use of scintillant beads made from either cerium-doped yttrium silicate (Y2SiO5:Ce) (hereafter referred to simply as yttrium silicate or YSi) or polyvinyltoluene (PVT) containing an organic scintillant such as PPO. Assays are carried out in aqueous buffers using radioisotopes such as 3H, 125I, 14C, 35S or 33P, that emit low-energy radiation, the energy of which is easily dissipated in an aqueous environment. For example, the electrons emitted by 3H have an average energy of only 6 keV and have a very short path length (xcx9c1 xcexcm) in water. If a molecule labelled with one of these isotopes is bound to the bead surface, either directly or via interaction with another molecule previously coupled to the bead, the emitted radiation will activate the scintillant and produce light. The amount of light produced, which is proportional to the amount of labelled molecules bound to the beads, can be measured conveniently with a liquid scintillation (LS) counter. If the labelled molecule is not attached to the bead surface, its radiation energy is absorbed by the surrounding aqueous solvent before it reaches the bead, and no light is produced. Thus, bound ligands give a scintillation signal, but free ligands do not, and the need for a time-consuming separation step, characteristic of conventional radioligand binding assays, is eliminated. The manipulations required in the assays are reduced to a few simple pipetting steps leading to better precision and reproducibility.
PCT WO 91/08489 (Packard Instrument Company Inc.) describes a support body for use in scintillation proximity radioimmunoassay, the support body being constructed of a scintillating material, having coupled to its surface a multiplicity of ligands such a antigens, antibodies, etc. capable of selectively binding a reactant of interest. Preferably the support bodies consist of yttrium silicate activated with an inorganic cerium salt such as the oxide, carbonate, or chloride.
WO 94/26413 concerns the study of cellular and biochemical processes in living cells or in components of cells. Specifically described are devices and methods for the study of cellular and biochemical processes, using the scintillation proximity principle.
The simplicity of the scintillation proximity format allows almost complete automation of assays using robotic sample processors and microtitre plate scintillation counters. Consequently, SPA technology is capable of high throughput, which is particularly valuable in the case of drug- or sample-screening assays. SP assays have been carried out routinely in 96-well microtitre plates which are counted 6 wells at a time in specially designed microtitre plate scintillation counters. The search for increasingly higher throughput has led the manufacturers of these counters to produce instruments capable of counting 12 wells at a time, thus doubling throughput. It has also seen the advent of 384-well plates, although at present these can still only be counted 12-wells at a time.
A problem associated with SPA is that of colour quenching, caused by the presence in the assay medium of coloured compounds that absorb the light emitted by the current SPA bead types. Colour quenching attenuates the signal, thereby decreasing signal to noise and hence the sensitivity. Many of the samples being screened by SPA assays are coloured and the majority of these are yellow or brown in colour and absorb light in the blue region of the visible spectrum. Both PVT- and Y2SiO5:Ce-based SPA beads emit light in the blue region (maximal emission normally in the range 350 nm-450 nm) and so are susceptible to this effect.
An alternative detection system suitable for use in low to ultra-low light level imaging applications in the biological and biomedical sciences is CCD (Charge Coupled Device) Detection which has been used, for example, in assays which involve chemiluminescent, bioluminescent and fluorescence detection. Applications include immunoassays (Hooper et al, J.Biolum.Chemilum., 9, 113-122, (1994)), and the analysis of specific fluorescent dye-labelled nucleic acids by hybridisation following electrophoretic separation of nucleic acid samples (EP 214713 to Astromed Ltd.). Ultra low-light imaging using CCD technology is quantitative and fast and the new generation of imaging instruments which use CCD cameras for detectors can image the whole of a plate at once and so have great potential for increasing sample throughput compared with microtitre well plate scintillation counters. Area imaging, i.e. the simultaneous imaging by CCD of all wells in a microtitre well plate is considered to be particularly advantageous when used in conjunction with high well-density plates containing 96, 384, 864, or more wells, since the time required to make measurements is significantly reduced compared with conventional scintillation counting techniques.
Imaging technology, in particular area imaging, has also been applied to isotopically labelled materials as an alternative to autoradiography. This approach has been most widely used in applications such as the quantitative analysis of proteins by 2-D gel electrophoresis (Patterson, et al, Biotechniques, 15(6), 1076, (1993)) and receptor localisation (Tang, et al, Biotechniques, 18(5), 886, (1995)). An imaging plate, coated with a radiation sensitive agent (e.g. strontium sulphide/samarium/cerium or barium fluorobromide/europium) is exposed to a radiolabelled sample and an image is formed due to radiation incident on the lanthanide metal coating of the plate. Following exposure, the image is read by means of an imaging plate reader.
CCD detection of SPA counts has also been reported (Englert, D., Society for Biomolecular Screening, Second Annual Conference Oct. 14-17, (1996), pp209-221) using PVT-based microspheres. However, the photon count from the SPA wells was not sensitive enough to enable usable results to be obtained, due to low light output of the beads, sub-optimal signal detection capability of the system, as well as quenching by coloured samples. For conventional scintillation counting, instruments can be calibrated to take into account colour quenching. However, in the case of CCD detection using conventional SPA beads and under normal assay conditions, the number of photons detected per disintegration was insufficient to enable determination of quenching levels and quench correction was not possible. To date there appear to be no reports of working assays in which sample detection and measurement was obtained using this technique.
The present invention seeks to overcome the dual problems of low sensitivity of current CCD-based detection as well as colour quench in conventional SPA bead technology. The invention provides use in a scintillation proximity test of a phosphor that has an emission maximum of 480 nm-900 nm, and of a charge coupled device for detecting radiation emitted by the phosphor.
A scintillation proximity test is a test in which a surface carrying a phosphor is contacted with a body of fluid containing a radioisotope. Part of the radioisotope becomes immobilised adjacent the surface; the remainder of the radioisotope remains dispersed or dissolved in the fluid. The mean free path of electrons or other particles or radiation resulting from radioactive disintegrations of the radioisotope are small relative to the dimensions of the body of fluid, whereby that part of the radioisotope immobilised adjacent the surface is capable of exciting phosphor carried by the surface, but that part of the radioisotope dispersed or dissolved in the fluid is generally too far from the surface to be capable of exciting phosphor carried by the surface.
The surface may be massive, as for example a wall of a vessel or wells of a multiwell or microtitre plate; or particulate, as for example threads or beads. The phosphor may be present as a coating applied on a pre-formed surface; or may be dispersed in or constitute or form part of the surface.
The test may be a chemical or biochemical assay, for example a competition assay such as an immunoassay or immunometric assay. Or the test may involve a study of living cells which are, or which become, attached to the surface carrying the phosphor. Any test system in which a radioisotope becomes partitioned between a solid phase and a liquid phase is in principle suitable for the method of the invention.
A radioisotope may be present in free form or combined form, e.g. as an atom or ion; this may be useful for example when it is desired to monitor the take up of the radioisotope by cells adhering to the surface carrying the phosphor. Or the radioisotope may be used to label an assay reagent; this may be useful for example when a labelled reagent is caused to compete with an unlabelled reagent for binding to another reagent immobilised on the surface carrying the phosphor.
A scintillation proximity test may be performed in a qualitative or more usually in a quantitative manner. For example, measurements may be performed in a static mode, as when the result of a competition assay is determined after a fixed time or at equilibrium. Alternatively a scintillation proximity assay may be performed in a dynamic mode, as when radiolabel uptake by cells is monitored in real time. WO 94/26413 describes a scintillating microtitre plate and methods for studying cellular processes in real time. The scintillating microtitre plate is marketed by Amersham Lifescience under the name Cytostar-T(trademark).
The fluid is generally an aqueous or other liquid. The radioisotope is preferably one which emits electrons having a mean free path up to 2000 xcexcm in aqueous media. These include isotopes commonly used in biochemistry such as 3H, 125I, 14C, 35S, 45Ca, 33P and 32P, but does not preclude the use of other radioisotopes such as 55Fe, 86Rb, 109Cd and 51Cr which also emit electrons within this range.
The scintillation proximity test is preferably performed in the wells of a multiwell plate e.g. a microtitre plate. The phosphor may be provided as beads dispensed into the wells of such a plate. Or the phosphor may be incorporated into the plate itself, either by direct incorporation into the plastic of the plate, or by coating, together with a binding agent. Examples of possible binding agents are calcium sulphate, as used in the manufacture of tic plates, and low-melting plastics such as polystyrene or copolymers of xcex1-methylstyrene and vinyltoluene. These devices may have 24, 96 or 384 wells as in existing plates or may have higher densities of wells such as 864, 1536, 2400 3456 or indeed any desired number. They can be used to perform cell-based or ligand binding assays in conjunction with CCD camera based imagers. The phosphor preferably has an emission maximum of 500 nm-700 nm, that is to say in the green or yellow or red region of the spectrum. The phosphors are stimulated by low energy electrons or other particles or radiation resulting from radioactive disintegrations of the radioisotope. These phosphors generally have a higher light output than PVT based SPA beads or Y2SiO5:Ce beads and enable SPA assays to be imaged successfully. Moreover, all sample wells of a microtitre well plate can be imaged simultaneously by means of CCD detection. The longer wavelength green or red emissions alleviate the colour quenching problem which is at its greatest in the blue region of the spectrum.
Some phosphors are commercially available for industrial applications, for example in cathode ray tube technology, lamp phosphors and X-ray phosphors. See for example Blasse and Grabmaier, Luminescent Materials, Springer-Verlag, Berlin, (1994) and U.S. Pat. No. 5,435,937. The preparation of charge-stabilised suspensions of small phosphor particles (e.g. yttrium oxysulphide-Eu3+, yttrium oxysulphide-Tb3+) and their coupling to antibodies to give immunoreactive conjugates has been described (Beverloo, et al, Cytometry, 13, 561-570 (1992)). The phosphor conjugates were used in immunocytochemical applications by binding to cells, followed by visualisation using a fluorescence microscope under UV light excitation. To date however, such phosphors have not been described for use in counting applications involving radioactive tracers, particularly SPA
There are many suitable phosphors that may be used. Some consist of an inorganic host material doped with an activator. Examples of such host materials are yttrium silicate, yttrium oxide, yttrium oxysulphide, yttrium aluminium gallium oxide (YAG), yttrium aluminium garnet, sodium yttrium fluoride (NaYF4), lanthanum fluoride, lanthanum oxysulphide, yttrium fluoride (YF3), yttrium gallate, gadolinium fluoride (GdF3), barium yttrium fluoride (BaYF5 or BaY2F8), gadolinium oxysulphide, zinc silicate, zinc sulphide and yttrium vanadate. The activator is generally a lanthanide or actinide moiety.
Other phosphors are organic chelates of lanthanide or actinide moieties. Light emission may be enhanced by combining or mixing the lanthanide or actinide chelate with a Lewis base enhancer such as for example an imido phosphorane. Examples of phosphors of this kind are described in EPA 556005. They may conveniently be used in solution or dispersion in polystyrene or other organic polymer.
The identity of the lanthanide or actinide moiety determines the emission wavelength of the phosphor. Preferred moieties are selected from terbium, europium, erbium, thulium, holmium, dysprosium, samarium, ytterbium, lutecium, gadolinium, uranium and uranyl UO2, generally in the form of +2 or +3 ions.